In order to study the virulence and biological characteristics of different isolates of grass carp reovirus (GCRV) type II, we researched two new isolates of genotype II GCRV, ZH180804 and CQ180701, which were isolated from diseased and healthy Ctenopharyngodon idella, respectively. The characteristics of cell culture, pathogenicity, genomic segment, gene sequence and phylogenetic differences of the two strains were compared and analyzed. The results showed that mortality rate of ZH180804 to C. idella and Gobiocypris rarus were 80%, 100%, and those of CQ180701 were 10%，0%, respectively. The data of mortality rate showed that ZH180804 is a virulent strain, and CQ180701 is a low virulent strain. Cell infection experiment showed the two isolates could proliferate in grass carp swim bladder cells (GSB), Gobiocypris rarus egg cells (GRE) and (G. rarus fin (GRF) cells, but did not cause cytopathic effect (CPE), and viral multiplication of ZH180804 strains was 1 000 times higher than that of CQ180701 strains. The banding patterns of two isolates in sodium dialkyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) were partially identical, but there were some differences between the segments of S7, S8, S9, S10 and S11. The sequence homology of the S7 and S11 genes between two viruses are 98% and 99%, respectively. Phylogenetic analysis indicated that isolates ZH180804 and CQ180701 belong to the same branch, and it means that they are very closely related. All results above indicate that two strains of GCRV type II isolated from diseased and healthy grass carp have more common characteristics, but their replication ability and pathogenicity are different.